Background: Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that is plays major roles ischemia/reperfusion (I/R) injury, heart failure and cardiac contractility. Although S1P is known to contribute to ischemic preconditioning (IPC), its mode of action remains unknown. Here, we hypothesized that IPC may induce alterations in mitochondrial function through alterations of mitochondrial S1P content.

Methods: Mice and rat hearts were  perfused on a Langendorff apparatus, and protocols for IPC and I/R applied. Mitochondria were isolated by density centrifugation; membrane potential and respiration were measured by an OROBOROS O2K-Oxygraph. Absolute S1P levels were determined by LC-MS/MS.  Elevation of whole body S1P in C57BL6 mice in vivo was achieved by treatment with the S1P lyase inhibitor 4′-deoxypyridoxine (DOP; 30mg/KG bodyweight/2 weeks).

Results: Isolated mitochondria from IPC hearts showed a 1.8-fold increase in S1P and higher oxygen consumption compared to time-controls. S1P content was dramatically decreased in mitochondria from I/R hearts in contrast to mitochondria from IPC hearts where it was 50% higher along with better mitochondrial function. Isolated mitochondria rapidly took up exogenous S1P in vitro and exhibited higher mitochondrial oxygen consumption. Mitochondria from DOP-treated mice exhibited higher S1P content and better respiration, whereas mitochondria from Sphk2 deficient mice had 2.5-fold reduced S1P content and diminished oxygen consumption that could be restored by exogenous S1P loading. In vitro, Ca2+ induced mitochondrial damage could be substantially attenuated by a 10-min S1P loading as measured by respiration and release of cytochrome c.

Conclusion: Cardioprotection by IPC may be in part mediated by mitochondrial S1P accumulation that boosts oxygen consumption and protects mitochondria against structural damage. Targeting mitochondrial S1P content may offer novel approaches to cardiac diseases where mitochondrial function is crucial to pathophysiology.